The long-range objective of this research is to elucidate the relationship between cell membrane structure and the cell membrane functions of solute transport and permeability, particularly as this relationship is expressed in a malignant cell, the Ehrlich mouse ascites tumor cell. The goal for this grant period is to define the action of probes which inhibit chloride transfer, prior to initiating biochemical investigations to identify membrane constituent(s) which mediate chloride movement. By focusing on chloride transfer, which is being studied extensively with physiologic and biochemical methods in human erythrocytes, we can learn about membrane differences between that cell and a rapidly proliferating mammalian eukaryotic cell. Studies will assess whether inhibitory probes interact specifically with a chloride transport system and will determine which probes will be most suitable for future biochemical labeling experiments. Experiments to determine the mechanism of action of furosemide will continue; this diuretic drug substantially inhibits one for one chloride exchange and its effect is partly irreversible. A number of other small chemical probes will be assessed for their effectiveness under different conditions, their specificity, reversibility, and mechanism of action. In order to characterize clearly the transfer process and the mechanism of probe action, procedures will be developed to investigate chloride flux in the absence of competing anions. This will allow determination of whether there is saturation behavior in the relationship between chloride flux and chloride concentration and the way in which probes affect this relationship; of whether or not there is competition of chloride and a probe for a common membrane site; and of the magnitude of chloride conductance (net flux) versus self-exchange and of the effects of a probe on each. The action of probes on transfer of ions like potassium and sulfate will be determined to help evaluate specificity.